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Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer’s LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experiments

Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer’s LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experiments

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P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real...

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... P2XRs, of which there are seven sub-types, are ATP-gated ion channels that are inducibly permeable to cations. With prolonged activation, P2RX7 channels become non-selectively permeable, resulting in the diffusion of high molecular weight molecules such as ATP; IL-1b and IL-18 release; large molecular weight dye uptake; K + efflux; Na + and Ca 2+ influx; membrane phosphatidylserine-flip; membrane blebbing and cell death (10)(11)(12)(13)(14). Most P2RXs are activated by ATP concentrations in the nanomolar to low micromolar range, but P2RX7 activation requires millimolar concentrations of eATP (15)(16)(17). ...
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Introduction Breast cancer affects two million patients worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP is degraded by eATPases. Given that eATP is toxic to cancer cells, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy. Methods TNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPases or specific antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively. Results In the present study, we observed dose-dependent declines of cell viability and increases in eATP of paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases, but not of the MCF-10A cell line. These effects were reversed by specific antagonists of P2RXs. Similar results, as those observed with eATPase inhibitors, were seen with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels. Conclusion These results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines, which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.
... MICROGLIA AND PD PATHOLOGY: POTASSIUM EFFLUX K + efflux is widely accepted as a consensus step for the assembly of the NLRP3 inflammasome scaffold [64][65][66][88][89][90][91][92] . This dependence has been attributed to the action of the protein NEK7 (never-inmitosis/NIMA-related kinase 7), which is recruited to the NLRP3 inflammasome complex and is crucial for its activation downstream of K + efflux 66,92,93 . ...
... NEK7 only operates outside of the mitosis portion of the cell cycle, meaning that the cells that are susceptible to inflammasome activation are limited to those in interphase 66 , and thus not all cells in a stimulated culture display NLRP3 inflammasome activity simultaneously. Elevated extracellular K + levels in the range of 50-130 mM have been reported to inhibit various mechanisms of NLRP3 inflammasome activation, both canonical and noncanonical 91,94 (Pike et al., in press), by eliminating the electrochemical gradient that drives K + efflux leading to NEK7 signaling. ...
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The pathology of Parkinson’s disease (PD) is characterized by α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration in the substantia nigra with collateral striatal dopamine signaling deficiency. Microglial NLRP3 inflammasome activation has been linked independently to each of these facets of PD pathology. The voltage-gated potassium channel Kv1.3, upregulated in microglia by α-synuclein and facilitating potassium efflux, has also been identified as a modulator of neuroinflammation and neurodegeneration in models of PD. Evidence increasingly suggests that microglial Kv1.3 is mechanistically coupled with NLRP3 inflammasome activation, which is contingent on potassium efflux. Potassium conductance also influences dopamine release from midbrain dopaminergic neurons. Dopamine, in turn, has been shown to inhibit NLRP3 inflammasome activation in microglia. In this review, we provide a literature framework for a hypothesis in which Kv1.3 activity-induced NLRP3 inflammasome activation, evoked by stimuli such as α-synuclein, could lead to microglia utilizing dopamine from adjacent dopaminergic neurons to counteract this process and fend off an activated state. If this is the case, a sufficient dopamine supply would ensure that microglia remain under control, but as dopamine is gradually siphoned from the neurons by microglial demand, NLRP3 inflammasome activation and Kv1.3 activity would progressively intensify to promote each of the three major facets of PD pathology: α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration. Risk factors overlapping to varying degrees to render brain regions susceptible to such a mechanism would include a high density of microglia, an initially sufficient supply of dopamine, and poor insulation of the dopaminergic neurons by myelin.
... Elevated extracellular K + is a well-known inhibitor of canonical NLRP3 inflammasome activation in mouse macrophages and human monocytes, as charge and concentration gradient-regulated K + efflux from the cell is usually, but not always, a signal converging from structurally diverse stimuli to trigger NLRP3 oligomerization leading to inflammasome scaffold assembly and IL-1β processing [16,[41][42][43][44][45][46]. Voltage-gated Kv1 potassium channels have been shown to be responsible for DA release in the brain in vivo [47], tying K + flux to DA release, so the ability of elevated extracellular K + to block different modes of NLRP3 inflammasome activation in human microglia was considered relevant to PD and was, therefore, also explored. ...
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Background Parkinson’s disease (PD) is characterized by the loss of nigral dopaminergic neurons leading to impaired striatal dopamine signaling, α-synuclein- (α-syn-) rich inclusions, and neuroinflammation. Degenerating neurons are surrounded by activated microglia with increased secretion of interleukin-1β (IL-1β), driven largely by the NLRP3 inflammasome. A critical role for microglial NLRP3 inflammasome activation in the progression of both dopaminergic neurodegeneration and α-syn pathology has been demonstrated in parkinsonism mouse models. Fibrillar α-syn activates this inflammasome in mouse and human macrophages, and we have shown previously that the same holds true for primary human microglia. Dopamine blocks microglial NLRP3 inflammasome activation in the MPTP model, but its effects in this framework, highly relevant to PD, remain unexplored in primary human microglia and in other in vivo parkinsonism models. Methods Biochemical techniques including quantification of IL-1β secretion and confocal microscopy were employed to gain insight into dopamine signaling-mediated inhibition of the NLRP3 inflammasome mechanism in primary human microglia and the SYN120 transgenic mouse model. Dopamine and related metabolites were applied to human microglia together with various inflammasome activating stimuli. The involvement of the receptors through which these catecholamines were predicted to act were assessed with agonists in both species. Results We show in primary human microglia that dopamine, l -DOPA, and high extracellular K ⁺ , but not norepinephrine and epinephrine, block canonical, non-canonical, and α-syn-mediated NLRP3 inflammasome-driven IL-1β secretion. This suggests that dopamine acts as an inflammasome inhibitor in human microglia. Accordingly, we provide evidence that dopamine exerts its inhibitory effect through dopamine receptor D1 and D2 (DRD1 and DRD2) signaling. We also show that aged mice transgenic for human C-terminally truncated (1–120) α-syn (SYN120 tg mice) display increased NLRP3 inflammasome activation in comparison to WT mice that is diminished upon DRD1 agonism. Conclusions Dopamine inhibits canonical, non-canonical, and α-syn-mediated activation of the NLRP3 inflammasome in primary human microglia, as does high extracellular K ⁺ . We suggest that dopamine serves as an endogenous repressor of the K ⁺ efflux-dependent microglial NLRP3 inflammasome activation that contributes to dopaminergic neurodegeneration in PD, and that this reciprocation may account for the specific vulnerability of these neurons to disease pathology.
... The application of P2X7R antagonists, such as A-438079 and A-804598, prevents both the current responses to ATP and the ATPinduced uptake of large cationic fluorescent dyes in cultured human microglia [57]. This process is related to several other immunological functions, especially in inflammasome activation [58,59]. Some other effects, such as activation of p38 MAP kinase [60,61], activation of phospholipase D [62,63], the production of reactive oxygen/nitrogen species [56], and killing of Mycobacterium tuberculosis [64,65] were also involved in the opening of large membrane pores. ...
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Macrophages are mononuclear phagocytes which derive either from blood-borne monocytes or reside as resident macrophages in peripheral (Kupffer cells of the liver, marginal zone macrophages of the spleen, alveolar macrophages of the lung) and central tissue (microglia). They occur as M1 (pro-inflammatory; classic) or M2 (anti-inflammatory; alternatively activated) phenotypes. Macrophages possess P2X7 receptors (Rs) which respond to high concentrations of extracellular ATP under pathological conditions by allowing the non-selective fluxes of cations (Na+, Ca2+, K+). Activation of P2X7Rs by still higher concentrations of ATP, especially after repetitive agonist application, leads to the opening of membrane pores permeable to ~900 Da molecules. For this effect an interaction of the P2X7R with a range of other membrane channels (e.g., P2X4R, transient receptor potential A1 [TRPA1], pannexin-1 hemichannel, ANO6 chloride channel) is required. Macrophage-localized P2X7Rs have to be co-activated with the lipopolysaccharide-sensitive toll-like receptor 4 (TLR4) in order to induce the formation of the inflammasome 3 (NLRP3), which then activates the pro-interleukin-1β (pro-IL-1β)-degrading caspase-1 to lead to IL-1β release. Moreover, inflammatory diseases (e.g., rheumatoid arthritis, Crohn’s disease, sepsis, etc.) are generated downstream of the P2X7R-induced upregulation of intracellular second messengers (e.g., phospholipase A2, p38 mitogen-activated kinase, and rho G proteins). In conclusion, P2X7Rs at macrophages appear to be important targets to preserve immune homeostasis with possible therapeutic consequences.
... Calcium signaling is a complex event that controls many cellular processes, including activation of NLRP3 inflammasome [69][70][71][72]. However, the precise mechanism of Ca 2+ associated with the NLRP3 inflammasome pathway is largely unclear. ...
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Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca²⁺ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.
... In general, Ca 2+ influx caused by the opening of the cell membrane channel is accompanied by K + efflux, serving as a counterion to Ca 2+ influx. Treatment with potassium on the extracellular side causes sustained calcium conduction from the extracellular side to the cytoplasmic side, rather than a transient calcium flux from intracellular stores, and thereby induces NLRP3 inflammasome activation in ATP-exposed J774A.1 macrophages, 38 suggesting that K + efflux and Ca 2+ influx cooperate to activate the assembly of NLRP3 inflammasome. However, contradictory evidence has demonstrated that Ca 2+ influx is not pivotal for NLRP3 inflammasome activation, as it occurs after NLRP3 inflammasome and caspase-1 activation. ...
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Chunteng Jiang,1,2 Santuan Xie,1 Guang Yang,3 Ningning Wang3 1Department of Internal Medicine, The Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning, People’s Republic of China; 2Department of Cardiology and Pneumology, University Medical Center of Göttingen, Georg-August-University of Göttingen, Göttingen, Lower Saxony, Germany; 3Department of Food Nutrition and Safety, School of Public Health, Dalian Medical University, Dalian, Liaoning, People’s Republic of ChinaCorrespondence: Guang Yang; Ningning Wang Email yg290@163.com; zkxwnn@dmu.edu.cnAbstract: Inflammation is an intricate biological response of body tissues to detrimental stimuli. Cardiovascular disease (CVD) is the leading cause of death worldwide, and inflammation is well documented to play a role in the development of CVD, especially atherosclerosis (AS). Emerging evidence suggests that activation of the NOD-like receptor (NLR) family and the pyridine-containing domain 3 (NLRP3) inflammasome is instrumental in inflammation and may result in AS. The NLRP3 inflammasome acts as a molecular platform that triggers the activation of caspase-1 and the cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin D (GSDMD). The cleaved GSDMD forms pores in the cell membrane and initiates pyroptosis, inducing cell death and the discharge of intracellular pro-inflammatory factors. Hence, the NLRP3 inflammasome is a promising target for anti-inflammatory therapy against AS. In this review, we systematically summarized the current understanding of the activation mechanism of NLRP3 inflammasome, and the pathological changes in AS involving NLRP3. We also discussed potential therapeutic strategies targeting NLRP3 inflammasome to combat AS.Keywords: cardiovascular disease, atherosclerosis, NLRP3 inflammasome, mechanisms, therapeutic strategies
... Another underlying signaling known to be associated with NAMP-activating NLRP3 inflammasome is the Ca 2+ influxmitochondrial ROS production pathway. 27,72 To examine whether these nanoparticles also follow this pathway in addition to the lysosomal disruption signaling, we first assessed calcium flux in iBMDMs treated with y = 0−400 series of sNPs as compared to only LPS-primed iBMDMs. To measure intracellular Ca 2+ levels, we stained the treated cells with a cell-permeant calcium indicator, Fluo-4AM, and analyzed them using either flow cytometry or confocal microscopy (Supplementary Figure S10a). ...
... We do not know exactly which membrane these NLRP3 cages reside; however TGN membrane localization of NLRP3 is visible before nigericin stimulation (Fig. 4), and additional NLRP3 may be recruited to the TGN upon nigericin treatment as suggested previously 9 . Because almost all NLRP3 activating stimuli converges to K + efflux 43,44 , it is reasonable that a drop in intracellular K + concentration could enhance charged interactions and TGN recruitment of NLRP3. ...
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The nucleotide-binding domain and leucine-rich-repeat (LRR) containing protein 3 with a pyrin domain (NLRP3) is emerging to be a critical intracellular inflammasome sensor of membrane integrity and a highly important clinical target against chronic inflammation. Here we report that the endogenous, stimulus-responsive form of full-length NLRP3 is a 12-16 mer double ring cage held together by LRR-LRR interactions with the pyrin domains shielded within the assembly to avoid premature activation. Surprisingly, this NLRP3 form is predominantly membrane localized, which is consistent with previously noted localization of NLRP3 at various membrane organelles. Structure-guided mutagenesis reveals that trans-Golgi network dispersion into vesicles, an early event observed for all NLRP3 activating stimuli, requires the double ring cages of NLRP3. Double ring-defective NLRP3 mutants further abolish inflammasome punctum formation, caspase-1 processing and cell death. Thus, unlike other inflammasome sensors that are monomeric when inactive, our data uncover a unique NLRP3 oligomer on membrane that is poised to sense diverse signals to induce inflammasome activation.
... There is substantial documentation in the scientific literature suggesting the role of Ca 2+ in inflammasome provoking. An increment in cellular Ca 2+ can evoke NLRP3 activation by processes that are still nebulous; on the other hand, calcium channel inhibition precludes NLRP3 stimulation [262,263]. Nilvadipine, which is an inhibitor of calcium channels in addition to being a documented Spleen tyrosine kinase (Syk) inhibitor, may be an alluring molecule to inhibit NLRP3 inflammasome stimulation. Treatment with Nilvadipine ameliorated the TBI-induced inflammatory response in aged mild traumatic brain injury (r-mTBI) animals and notably augmented spatial memory. ...
... There is substantial documentation in the scientific literature suggesting the role of Ca 2+ in inflammasome provoking. An increment in cellular Ca 2+ can evoke NLRP3 activation by processes that are still nebulous; on the other hand, calcium channel inhibition precludes NLRP3 stimulation [262,263]. Nilvadipine, which is an inhibitor of calcium channels in addition to being a documented Spleen tyrosine kinase (Syk) inhibitor, may be an alluring molecule to inhibit NLRP3 inflammasome stimulation. Treatment with Nilvadipine ameliorated the TBI-induced inflammatory response in aged mild traumatic brain injury (r-mTBI) animals and notably augmented spatial memory. ...
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Nucleotide-binding oligomerization domain NOD-like receptors (NLRs) are conserved cytosolic pattern recognition receptors (PRRs) that track the intracellular milieu for the existence of infection, disease-causing microbes, as well as metabolic distresses. The NLRP3 inflammasome agglomerates are consequent to sensing a wide spectrum of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Certain members of the NLR family have been documented to lump into multimolecular conglomerates called inflammasomes, which are inherently linked to stimulation of the cysteine protease caspase-1. Following activation, caspase-1 severs the proinflammatory cytokines interleukin (IL)-1β and IL-18 to their biologically active forms, with consequent commencement of caspase-1-associated pyroptosis. This type of cell death by pyroptosis epitomizes a leading pathway of inflammation. Accumulating scientific documentation has recorded overstimulation of NLRP3 (NOD-like receptor protein 3) inflammasome involvement in a wide array of inflammatory conditions. IL-1β is an archetypic inflammatory cyto-kine implicated in multiple types of inflammatory maladies. Approaches to impede IL-1β's actions are possible, and their therapeutic effects have been clinically demonstrated; nevertheless, such strategies are associated with certain constraints. For instance, treatments that focus on systemically negating IL-1β (i.e., anakinra, rilonacept, and canakinumab) have been reported to result in an escalated peril of infections. Therefore, given the therapeutic promise of an NLRP3 inhibitor, the concerted escalated venture of the scientific sorority in the advancement of small molecules focusing on direct NLRP3 inflammasome inhibition is quite predictable.